Beta-carbolines as positive allosteric modulators of the human serotonin receptor 2c (5-ht2c)

ABSTRACT

Disclosed are novel small molecules, methods of synthesis of the small molecules, and uses of the small molecules for modulating activity of the human serotonin receptor 2C (5-HT2c), preferably selectively. The small molecules have a substituted beta-carboline core structure, which optionally may be saturated at one or more bonds to provide a dihydro-beta-carboline core or a tetrahydro-beta-carboline core. The small molecules may be administered to treat and/or prevent diseases, disorders, and/or conditions associated with human serotonin receptor 2C (5-HT2c) including psychiatric, mental, and/or neurological diseases, disorders, and conditions such as cognitive impairment, addiction, and obsessive compulsive disorder. The disclosed small molecules also may be administered to treat and/or prevent obesity, for example, via appetite suppression.

CROSS-REFERENCED TO RELATED PATENT APPLICATIONS

The present application is the U.S. National Stage Entry ofInternational Application PCT/US2019/054337, filed Oct. 2, 2019, whichclaims the benefit of priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/740,084, filed Oct. 2, 2018, thecontents of which are incorporated herein by reference in theirentireties.

BACKGROUND

The field of the invention relates to novel small molecules, methods ofsynthesis of the small molecules, and uses of the small molecules formodulating activity of the human serotonin receptor 2C (5-HT_(2c)). Thesmall molecules have a substituted beta-carboline core structure, whichoptionally may be saturated at one or more bonds to provide adihydro-beta-carboline core or a tetrahydro-beta-carboline core. Thesmall molecules may be administered to treat and/or prevent diseases,disorder, and condition associated with human serotonin receptor 2C(5-HT_(2c)) activity including psychiatric, mental, and/or neurologicaldiseases, disorders, and conditions such as cognitive impairment,addiction, and obsessive compulsive disorder.

Serotonin receptors, otherwise referred to as 5-hydroxytryptaminereceptors or 5-HT receptors, are a group of G protein-coupled receptorand ligand-gated ion channels found in the central and peripheralnervous systems. (See Hoyer et al., (1994). “International Union ofPharmacology classification of receptors for 5-hydroxytryptamine(Serotonin)”. Pharmacol. Rev. 46 (2): 157-203. PMID 7938165; Frazer etal., “Chapter 13: Serotonin Receptors”. In Siegel et al. BasicNeurochemistry: Molecular, Cellular, and Medical Aspects. Philadelphia:Lippincott-Raven. pp. 263-292; and Beliveau et al. (2017-01-04). “AHigh-Resolution In Vivo Atlas of the Human Brain's Serotonin System”.Journal of Neuroscience. 37 (1): 120-128.doi:10.1523/jneurosci.2830-16.2016; the contents of which areincorporated herein by reference in their entireties). Serotoninreceptors mediate excitatory neurotransmission and inhibitoryneurotransmission. The serotonin receptors, as they name indicates, areactivated by the neurotransmitter serotonin, which is their naturalligand. Serotonin receptors modulate the release of manyneurotransmitters, including glutamate, GABA, dopamine,epinephrine/norepinephrine, and acetylcholine, as well as many hormones,including oxytocin, prolactin, vasopressin, cortisol, corticotropin, andsubstance P, among others. The serotonin receptors influence variousbiological and neurological processes such as aggression, anxiety,cognition, learning, memory, mood, appetite, nausea, sleep, andthermoregulation. (See Nichols et al., (May 2008). “Serotoninreceptors.” Chem. Rev. 108(5): 1614-41; the content of which isincorporate herein by reference in its entirety).

The 5-HT₂ family of serotonin receptors mediates excitatoryneurotransmission via a G_(q)/G₁₁-protein coupled molecular mechanismthat increases cellular levels of inositol triphosphate (IP₃) anddiacylglycerol (DAG). The 5-HT-2 family includes three members:5-HT_(2a), 5-HT_(2b), and 5-HT_(2c). The 5-HT_(2a) receptor modulatesaddiction, anxiety, appetite, cognition, imagination, learning, memory,mood, perception, sexual behavior, sleep, thermoregulation, andvasoconstriction. The 5-HT_(2b) receptor modulates anxiety, appetite,cardiovascular function, gastrointestinal motility, sleep, andvasoconstriction. The 5-HT_(2c) receptor modulates addiction, anxiety,appetite, gastrointestinal motility, locomotion, mood, penile erection,sexual behavior, sleep, thermoregulation, and vasoconstriction. Notably,the 5-HT_(2a), 5-HT_(2b), and 5-HT_(2c) exhibit differing affinities foragonists and antagonists and may function as heteroreceptors for ligandsother than serotonin. For example, 5-HT_(2c) receptor is aheteroreceptor for norepinephrine and dopamine.

Here, we disclose novel substituted beta-carboline compounds whichfunction as positive allosteric modulators of the human serotoninreceptor 2C (5-HT_(2c)). The disclosed small molecules preferably actselectively as positive allosteric modulators of the human serotoninreceptor 2C (5-HT_(2c)), relative to other 5-HT receptors such as5-HT_(2a), 5-HT_(2b). As such, the small molecules may be administeredto treat and/or prevent diseases, disorder, and condition associatedwith human serotonin receptor 2C (5-HT_(2c)) including psychiatric,mental, and/or neurological diseases, disorders, and conditions such ascognitive impairment, addiction, and obsessive compulsive disorder.

SUMMARY

Disclosed are novel small molecules, methods of synthesis of the smallmolecules, and uses of the small molecules for modulating activity ofthe human serotonin receptor 2C (5-HT_(2c)), preferably selectively. Thesmall molecules have a substituted beta-carboline core structure, whichoptionally may be saturated at one or more bonds to provide adihydro-beta-carboline core or a tetrahydro-beta-carboline core. Thesmall molecules may be administered to treat and/or prevent diseases,disorders, and/or conditions associated with human serotonin receptor 2C(5-HT_(2c)) including psychiatric, mental, and/or neurological diseases,disorders, and conditions such as cognitive impairment, addiction, andobsessive compulsive disorder (OCD). The disclosed small molecules alsomay be administered to treat and/or prevent obesity, for example, viaappetite suppression.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . Locomotor Activity: AJC-61 blocked amphetamine-induced increasein locomotor activity in mice. The effect of AJC-61 (2 mg/kg) onamphetamine-induced (2.5 mg/kg) increase in locomotor activity in mice.Data are presented as group means±S.E.M. for eight successive 15 minuteintervals. *** p<0.001: significant increase in LMA versusVehicle+Vehicle; ###p<0.001: significant decrease in LMA versusVehicle+AJC-61 (2 mg/kg)+amphetamine (2.5 mg/kg).

FIG. 2 . Locomotor Activity: AJC-61 blocked PCP-induced increase inlocomotor activity in mice. The effect of AJC-61 (2 mg/kg) onPCP-induced (10 mg/kg) increase in locomotor activity in mice. Data arepresented as group means S.E.M. for eight successive 15 minuteintervals. *** p<0.001: significant increase in LMA versusVehicle+Vehicle; ###p<0.001: significant decrease in LMA versusVehicle+AJC-61 (2 mg/kg)+PCP (10 mg/kg). sc-subchronic.

FIG. 3 . Novel Object Recognition: Discrimination index (DI) for maleC57BL/6J mice treated with Vehicle, PCP (10 mg/kg), and PCP+AJC-61 (2mg/kg). Data are shown as mean S.E.M. of exploration time (s) n=10 miceper group. Significant differences were observed in the DI; ***p<0.001—significant decrease in DI in Vehicle versus PCP group.###p<0.001—significant increase in DI in PCP versus PCP+AJC-61, one-wayANOVA followed by post-hoc Bonferroni.

FIG. 4 . Social Interaction: Effect of Vehicle, PCP (10 mg/kg: i.p., 7days: b.i.d.), and PCP+AJC-61 (2 mg/kg), on social behaviors (sniffling,following, and avoiding) and interaction with inanimate object (objectexploration), in a 10-minute social interaction (SI) paradigm. Data wereanalyzed by one-way ANOVA comparing the effect of drug treatment on eachbehavior and are shown as mean±S.E.M. of time (s); n=10 pairs of miceper group. Sniffing—*** p, 0.001: Significant reduction for PCP versusVehicle group; ###p<0.001; Significant increase for PCP+AJC-61 versusPCP group. Following—No significant change, although there wasnon-significant reduction in following in the PCP versus Vehicle group.Avoiding—*** p<0.001: Significant increase for PCP versus Vehicle group;###p<0.001: Significant decrease for PCP+AJC-61 versus PCP group. ObjectExploration—No significant change, although there was a non-significantincrease in object exploration in the PCP versus Vehicle group.

FIG. 5 . Porsolt Forced Swim Test (FST). Effect of Vehicle, PCP (10mg/kg; i.p., 7 days; b.i.d.), and PCP+AJC-61 (2 mg/kg), in a 6-minPorsolt Forced Swim Test (FST). Data were analyzed by one-way ANOVAcomparing the effect of drug treatment on each behavior and are shown asmean S.E.M. of time (s): n=12 pairs of mice per group. *** p<0.001:Significant increase in immobility time for PCP versus Vehicle group;###p<0.001: Significant decrease in immobility time for PCP+AJC-16versus PCP group.

FIG. 6 . Marble Burying Task (MBT): Effect of Vehicle, PCP (10 mg/kg;i.p., 7 days; b.i.d.), and PCP+AJC-61 (2 mg/kg) in a 30-minute MarbleBurying Task (MBT) paradigm. Data were analyzed by one-way ANOVAcomparing the effect of drug treatment on each behavior and are shown asmean S.E.M. of time (s): n=12 pairs of mice per group. *** p<0.001:Significant increase in the number of marbles buried for PCP versusVehicle group; ###p<0.001: Significant decrease in the number of marblesburied for PCP+AJC-16 versus PCP group.

FIG. 7 . Nestlet Shredding (NS): Effect of Vehicle, PCP (10 mg/kg; i.p.,7 days; b.i.d.), and PCP+AJC-61 (2 mg/kg) in a 30-minute NestletShredding (NS) paradigm. Data were analyzed by one-way ANOVA comparingthe effect of drug treatment on each behavior and are shown asmean±S.E.M. of time (s): n=10 pairs of mice per group. *** p<0.001:Significant increase in the percent nestlet shredded for PCP versusVehicle group; ###p<0.001: Significant decrease in the percent nestletshredded for PCP+AJC-16 versus PCP group.

FIG. 8 . Novel Object Recognition: Effect on discrimination index (DI)of Vehicle and AJC-61 (2 mg/kg) on 2.5 month old mice and 22-month oldmice Data are shown as mean S.E.M. of exploration time (s): n=14 pairsof mice per group. Significant difference were observed in the DI: ***p<0.001: Significant decrease in the DI for 22-month old mice versus 2.5month old mice; ###p<0.001: Significant increase in the DI for 22-monthold mice treated with AJC-61 versus Vehicle.

DETAILED DESCRIPTION

The disclosed subject matter further may be described utilizing terms asdefined below.

Unless otherwise specified or indicated by context, the terms “a” “an”,and “the” mean “one or more.” For example, “a modulator of humanserotonin receptor 2C (5-HT₂c) activity” should be interpreted to mean“one or more modulators of human serotonin receptor 2C (5-HT₂c)activity.”

As used herein, “about”, “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of the term which are not clear to persons of ordinaryskill in the art given the context in which it is used, “about” and“approximately” will mean plus or minus ≤10% of the particular term and“substantially” and “significantly” will mean plus or minus >10% of theparticular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising.” The terms “comprise”and “comprising” should be interpreted as being “open” transitionalterms that permit the inclusion of additional components further tothose components recited in the claims. The terms “consist” and“consisting of” should be interpreted as being “closed” transitionalterms that do not permit the inclusion additional components other thanthe components recited in the claims. The term “consisting essentiallyof” should be interpreted to be partially closed and allowing theinclusion only of additional components that do not fundamentally alterthe nature of the claimed subject matter.

The terms “subject,” “patient,” and “individual” may be usedinterchangeably herein. A subject may be a human subject. A subject mayrefer to a human subject having or at risk for acquiring a disease,disorder, or condition, that is associated with human serotonin receptor2C (5-HT₂c) activity. In specific embodiments, the disease, disorder, orcondition is a psychiatric, mental, and/or neurological disease,disorder, or condition. Psychiatric, mental, and/or neurologicaldisease, disorders, or conditions may include, but are not limited to,cognitive impairment, addiction, and obsessive compulsive disorder(OCD). The disclosed small molecules also may be administered to treatand/or prevent obesity, for example, via appetite suppression, where thetreated and/or prevented disorder or condition is an eating disorderand/or obesity.

As used herein, the term “modulate” means decreasing or inhibitingactivity and/or increasing or augmenting activity. For example,modulating human serotonin receptor 2C (5-HT₂c) activity may meanincreasing or augmenting human serotonin receptor 2C (5-HT₂c) activityand/or decreasing or inhibiting human serotonin receptor 2C (5-HT₂c)activity. Preferably, the disclosed novel molecules increase or augmenthuman serotonin receptor 2C (5-HT₂c) activity as positive allostericligands or agonists.

As used herein, the phrase “effective amount” shall mean that drugdosage that provides the specific pharmacological response for which thedrug is administered in a significant number of patients in need of suchtreatment. An effective amount of a drug that is administered to aparticular patient in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

New Chemical Entities

New chemical entities and uses for chemical entities are disclosedherein. The chemical entities may be described using terminology knownin the art and further discussed below.

As used herein, an asterisk “*” or a plus sign “+” may be used todesignate the point of attachment for any radical group or substituentgroup.

The term “alkyl” as contemplated herein includes a straight-chain orbranched alkyl radical in all of its isomeric forms, such as a straightor branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to hereinas C1-C12 alkyl, C1-C10-alkyl, and C1-C6-alkyl, respectively.

The term “alkylene” refers to a diradical of an alkyl group (e.g.,—(CH₂)_(n)— where n is an integer such as an integer between 1 and 20).An exemplary alkylene group is —CH₂CH₂—.

The term “haloalkyl” refers to an alkyl group that is substituted withat least one halogen. For example, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CF₂CF₃,and the like.

The term “heteroalkyl” as used herein refers to an “alkyl” group inwhich at least one carbon atom has been replaced with a heteroatom(e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxy”group.

The term “alkenyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon double bond, suchas a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms,referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl,respectively.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms,referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl,respectively.

The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic,or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8,or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derivedfrom a cycloalkane. Unless specified otherwise, cycloalkyl groups areoptionally substituted at one or more ring positions with, for example,alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino,amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano,cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. Incertain embodiments, the cycloalkyl group is not substituted, i.e., itis unsubstituted.

The term “cycloalkylene” refers to a cycloalkyl group that isunsaturated at one or more ring bonds.

The term “partially unsaturated carbocyclyl” refers to a monovalentcyclic hydrocarbon that contains at least one double bond between ringatoms where at least one ring of the carbocyclyl is not aromatic. Thepartially unsaturated carbocyclyl may be characterized according to thenumber of ring carbon atoms. For example, the partially unsaturatedcarbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, andaccordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 memberedpartially unsaturated carbocyclyl, respectively. The partiallyunsaturated carbocyclyl may be in the form of a monocyclic carbocycle,bicyclic carbocycle, tricyclic carbocycle, bridged carbocycle,spirocyclic carbocycle, or other carbocyclic ring system. Exemplarypartially unsaturated carbocyclyl groups include cycloalkenyl groups andbicyclic carbocyclyl groups that are partially unsaturated. Unlessspecified otherwise, partially unsaturated carbocyclyl groups areoptionally substituted at one or more ring positions with, for example,alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino,amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano,cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. Incertain embodiments, the partially unsaturated carbocyclyl is notsubstituted, i.e., it is unsubstituted.

The term “aryl” is art-recognized and refers to a carbocyclic aromaticgroup. Representative aryl groups include phenyl, naphthyl, anthracenyl,and the like. The term “aryl” includes polycyclic ring systems havingtwo or more carbocyclic rings in which two or more carbons are common totwo adjoining rings (the rings are “fused rings”) wherein at least oneof the rings is aromatic and, e.g., the other ring(s) may becycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. Unlessspecified otherwise, the aromatic ring may be substituted at one or morering positions with, for example, halogen, azide, alkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl,carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide,ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties,—CF₃, —CN, or the like. In certain embodiments, the aromatic ring issubstituted at one or more ring positions with halogen, alkyl, hydroxyl,or alkoxyl. In certain other embodiments, the aromatic ring is notsubstituted, i.e., it is unsubstituted. In certain embodiments, the arylgroup is a 6-10 membered ring structure.

The terms “heterocyclyl” and “heterocyclic group” are art-recognized andrefer to saturated, partially unsaturated, or aromatic 3- to 10-memberedring structures, alternatively 3- to 7-membered rings, whose ringstructures include one to four heteroatoms, such as nitrogen, oxygen,and sulfur. The number of ring atoms in the heterocyclyl group can bespecified using 5 Cx-Cx nomenclature where x is an integer specifyingthe number of ring atoms. For example, a C3-C7 heterocyclyl group refersto a saturated or partially unsaturated 3- to 7-membered ring structurecontaining one to four heteroatoms, such as nitrogen, oxygen, andsulfur. The designation “C3-C7” indicates that the heterocyclic ringcontains a total of from 3 to 7 ring atoms, inclusive of any heteroatomsthat occupy a ring atom position.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines (e.g., mono-substituted amines ordi-substituted amines), wherein substituents may include, for example,alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

The terms “alkoxy” or “alkoxyl” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxy groups include methoxy, ethoxy, tert-butoxy andthe like.

An “ether” is two hydrocarbons covalently linked by an oxygen.Accordingly, the substituent of an alkyl that renders that alkyl anether is or resembles an alkoxyl, such as may be represented by one of—O-alkyl, —O-alkenyl, —O-alkynyl, and the like.

The term “carbonyl” as used herein refers to the radical —C(O)—.

The term “oxo” refers to a divalent oxygen atom —O—.

The term “carboxamido” as used herein refers to the radical —C(O)NRR′,where R and R′ may be the same or different. R and R′, for example, maybe independently alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl,heteroaryl, or heterocyclyl.

The term “carboxy” as used herein refers to the radical —COOH or itscorresponding salts, e.g. —COONa, etc.

The term “amide” or “amido” or “amidyl” as used herein refers to aradical of the form —R¹C(O)N(R²)—, —R¹C(O)N(R²)R³—, —C(O)NR²R³, or—C(O)NH₂, wherein R¹, R² and R³, for example, are each independentlyalkoxy, alkyl, alkenyl, alkynyl, amino, aryl, arylalkyl, carbamate,cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl,heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S” or “+” or “−” depending on the configuration ofsubstituents around the stereogenic carbon atom and or the opticalrotation observed. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers includeenantiomers and diastereomers. Mixtures of enantiomers or diastereomersmay be designated (±)” in nomenclature, but the skilled artisan willrecognize that a structure may denote a chiral center implicitly. It isunderstood that graphical depictions of chemical structures, e.g.,generic chemical structures, encompass all stereoisomeric forms of thespecified compounds, unless indicated otherwise. Also contemplatedherein are compositions comprising, consisting essentially of, orconsisting of an enantiopure compound, which composition may comprise,consist essential of, or consist of at least about 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a givencompound (e.g., at least about 99% of an R enantiomer of a givencompound).

Beta-Carboline Compounds and Uses Thereof for Modulating 5-HT₂, ReceptorActivity

The compounds disclosed herein may be referred to as beta-carbolinecompounds and in particular, substituted beta-carboline compounds.Optionally, the disclosed compounds may be saturated at one or morebonds to form dihydro-beta-carboline compounds ortetrahydro-beta-carboline compounds.

In some embodiments, the disclosed compound may be described ascompounds or a salts or solvates thereof having a Formula I:

where:

-   -   n is 0 or 1;    -   X is CH₂, NH, or O;    -   R¹ is selected from hydrogen, hydroxyl, alkyl (e.g. methyl),        alkoxy (e.g., methoxy), halo (e.g., fluoro, chloro, bromo,        iodo), haloalkyl (e.g., trifluoromethyl), amino, alkylamino, and        cyano;    -   R² is hydrogen, alkyl, or a 3-7 membered carbocycle or        heterocycle which is saturated or unsaturated at one or more        bonds and which heterocycle includes one or more heteroatoms        selected from N, O, and S, optionally which carbocycle or        heterocycle is substituted to include one or more non-hydrogen        substituents, which non-hydrogen substituents optionally are        selected from alkyl (e.g. methyl), alkoxy (e.g., methoxy), halo        (e.g., fluoro, chloro, bromo, iodo), haloalkyl (e.g.,        trifluoromethyl), hydroxyl, phenyl, amino, and carbonyl.    -   R³ is present or absent, and when R³ is present, R³ is selected        from selected from hydrogen, alkyl (e.g., methyl), alkenyl (e.g.        propenyl), and alkyl-alkoxy (e.g., propanyl-methoxy).

In some embodiments, the disclosed compounds may have a Formula Ia:

In some embodiments, the disclosed compound have a Formula Ib:

In the disclosed compounds, in some embodiments, R¹ may be selected fromhydrogen, alkyl (e.g., methyl), alkyoxy (e.g., methoxy), and halo (e.g.,chloro).

In the disclosed compounds, in some embodiments R¹ is selected from:

The compound of claim 1, wherein R² is selected from phenyl, cyclohexyl,pyridinyl (e.g., N-pyridinyl, pyridin-2-yl, pyridin-3-yl, orpyridin-4-yl), pyrimidinyl (e.g., pyrimidin-1-yl, pyrimidin-2-yl,pyrimidin-4-yl, or pyrimidin-5-yl), imidazolyl (e.g., imidazole-1-yl,imidazol-2-yl, or imidazole-4-yl), 1-methylimidazolyl (e.g.,1-methylimidazol-2-yl, 1-methylimidazol-3-yl, 1-methylimidazol-4-yl, or1-methylimidazol-5-yl), piperidinyl (e.g., piperidin-2-yl, orpiperidin-4-yl), 1-methyl-piperidinyl (1-methyl-piperidin-2-yl,1-methyl-piperidin-3-yl, 1-methyl-piperidin-4-yl), piperazinyl (e.g.,piperazin-1-yl, piperazin-2-yl, or piperazin-4-yl), 1-methyl-piperazinyl(e.g., 1-methyl-piperazin-2-yl, 1-methyl-piperazin-3-yl, or1-methyl-piperazin-4-yl), tetrahydropyranyl (e.g., tetrahydropyran-2-yl,tetrahydropyran-3-yl, or, tetrahydropyran-4-yl), and morpholinyl (e.g.,morpholin-2-yl, morpholin-3-yl, or morpholin-4-yl).

In the disclosed compounds, in some embodiments —(X)_(n)—R² is selectedfrom:

As noted, the compounds disclosed herein, including the substitutedbeta-carboline compounds discussed above may have several chiralcenters, and stereoisomers, epimers, and enantiomers are contemplated.The compounds may be optically pure with respect to one or more chiralcenters (e.g., some or all of the chiral centers may be completely inthe S configuration; some or all of the chiral centers may be completelyin the R configuration; etc.). Additionally or alternatively, one ormore of the chiral centers may be present as a mixture of configurations(e.g., a racemic or another mixture of the R configuration and the Sconfiguration). Compositions comprising substantially purifiedstereoisomers, epimers, or enantiomers, or analogs or derivativesthereof are contemplated herein (e.g., a composition comprising at leastabout 90%, 95%, 99% or 100% pure stereoisomer, epimer, or enantiomer.)As used herein, formulae which do not specify the orientation at one ormore chiral centers are meant to encompass all orientations and mixturesthereof.

Human Serotonin Receptor 2C (5-HT_(2c)) Activity Modulation

The compounds disclosed herein preferably modulate human serotoninreceptor 2C (5-HT₂c) activity and may be administered to a subject inneed thereof to modulate 5-HT_(2c) receptor activity. Modulation mayinclude activating or increasing human serotonin receptor 2C (5-HT₂c)activity. Modulation also may include inhibiting or decreasing humanserotonin receptor 2C (5-HT₂c) activity.

5-HT_(2c) receptor activity may be assessed utilizing methods known inthe art and the methods disclosed herein, including the methodsdisclosed in the Examples provided herein. In some embodiments, thecompounds decrease or increase 5-HT₂, activity relative to a control(e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more (orwithin a range bounded by any of these values)). In other embodiments,the compounds activate the 5-HT_(2c) receptor greater than about 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, relative to a control.In other embodiments, the compounds activate the 5-HT_(2c) receptor witha maximum activation (E_(max)) greater than about 100%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1100%, 1200%, 1300%, 1400%,or 1500% (or within a range bounded by any of these values). In otherembodiments, an EC₅₀ value for the compound in regard to activation ofthe 5-HT_(2c) receptor may be determined and preferably the compound hasan EC₅₀ value of less than about 10 μM, 5 μM, or 1 μM, 0.5 μM, 0.1 μM,0.05 μM, 0.01 μM, 0.005 μM, or 0.001 μM (or within a range bounded byany of these values). In other embodiments, K_(i) value for the compoundin regard to activation of the 5-HT_(2c) receptor may be determined andpreferably the compound has an K_(i) value of less than about 10 μM, 5μM, or 1 μM, 0.5 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, or 0.001 μM (orwithin a range bounded by any of these values).

In some embodiments, the compounds disclosed herein do not bind toand/or activate or inhibit the 5-HT_(2a) receptor or the 5-HT_(2b)receptor or any other receptor other than the 5-HT_(2c) receptor. If thecompounds bind to and/or activate the 5-HT_(2a) receptor or the 5-HT₂breceptor or any other receptor, preferably the compounds have an EC₅₀for the 5-HT_(2a) receptor or the 5-HT_(2b) receptor or any otherreceptor that is greater than about 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, or 1000 μM If thecompounds bind to and/or activate the 5-HT_(2a) receptor or the5-HT_(2b) receptor or any other receptor, preferably the compounds havea K_(i), for the 5-HT_(2a) receptor or the 5-HT_(2b) receptor or anyother receptor that is greater than about 0.01 μM, 0.05 μM, 0.1 μM, 0.5μM, 1 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, or 1000 μM.

Pharmaceutical Compositions and Methods of Administration

The compounds employed in the compositions and methods disclosed hereinmay be administered as pharmaceutical compositions and, therefore,pharmaceutical compositions incorporating the compounds are consideredto be embodiments of the compositions disclosed herein. Suchcompositions may take any physical form which is pharmaceuticallyacceptable; illustratively, they can be orally administeredpharmaceutical compositions. Such pharmaceutical compositions contain aneffective amount of a disclosed compound, which effective amount isrelated to the daily dose of the compound to be administered. Eachdosage unit may contain the daily dose of a given compound or eachdosage unit may contain a fraction of the daily dose, such as one-halfor one-third of the dose. The amount of each compound to be contained ineach dosage unit can depend, in part, on the identity of the particularcompound chosen for the therapy and other factors, such as theindication for which it is given. The pharmaceutical compositionsdisclosed herein may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing well known procedures.

The compounds for use according to the methods of disclosed herein maybe administered as a single compound or a combination of compounds. Forexample, a compound that modulates the 5-HT_(2c) receptor activity maybe administered as a single compound or in combination with anothercompound that modulates the 5-HT_(2c) receptor activity or that has adifferent pharmacological activity.

As indicated above, pharmaceutically acceptable salts of the compoundsare contemplated and also may be utilized in the disclosed methods. Theterm “pharmaceutically acceptable salt” as used herein, refers to saltsof the compounds which are substantially non-toxic to living organisms.Typical pharmaceutically acceptable salts include those salts preparedby reaction of the compounds as disclosed herein with a pharmaceuticallyacceptable mineral or organic acid or an organic or inorganic base. Suchsalts are known as acid addition and base addition salts. It will beappreciated by the skilled reader that most or all of the compounds asdisclosed herein are capable of forming salts and that the salt forms ofpharmaceuticals are commonly used, often because they are more readilycrystallized and purified than are the free acids or bases.

Acids commonly employed to form acid addition salts may includeinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suitablepharmaceutically acceptable salts may include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide,acetate, propionate, decanoate, caprylate, acrylate, formate,hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleat-, butyne-.1,4-dioate, hexyne-1,6-dioate, benzoate,chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate,phthalate, xylenesulfonate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate,tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Bases useful in preparing such salts includesodium hydroxide, potassium hydroxide, ammonium hydroxide, potassiumcarbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate,calcium hydroxide, calcium carbonate, and the like.

The particular counter-ion forming a part of any salt of a compounddisclosed herein is may not be critical to the activity of the compound,so long as the salt as a whole is pharmacologically acceptable and aslong as the counterion does not contribute undesired qualities to thesalt as a whole. Undesired qualities may include undesirably solubilityor toxicity.

Pharmaceutically acceptable esters and amides of the compounds can alsobe employed in the compositions and methods disclosed herein. Examplesof suitable esters include alkyl, aryl, and aralkyl esters, such asmethyl esters, ethyl esters, propyl esters, dodecyl esters, benzylesters, and the like. Examples of suitable amides include unsubstitutedamides, monosubstituted amides, and disubstituted amides, such as methylamide, dimethyl amide, methyl ethyl amide, and the like.

In addition, the methods disclosed herein may be practiced using solvateforms of the compounds or salts, esters, and/or amides, thereof. Solvateforms may include ethanol solvates, hydrates, and the like.

The pharmaceutical compositions may be utilized in methods of treatingand/or preventing a disease, disorder, or condition associated with5-HT_(2c) receptor activity. For example, the pharmaceuticalcompositions may be utilized to treat patients having or at risk foracquiring a psychiatric disease or disorder or condition. Suitablepatients include, for example mammals, such as humans and non-humanprimates (e.g., chimps) or other mammals (e.g., dogs, cats, horses,rats, and mice). Suitable human patients may include, for example, thosewho have previously been determined to be at risk of having ordeveloping a psychiatric disease, disorder, or condition associate with5-HT_(2c) receptor activity.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors can be considered by the attending diagnostician, suchas: the species of the subject; its size, age, and general health; thedegree of involvement or the severity of the disease or disorderinvolved; the response of the individual subject; the particularcompound administered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A typical daily dose may contain from about 0.01 mg/kg to about 100mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about0.1 mg/kg to about 25 mg/kg) of each compound used in the present methodof treatment.

Compositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 500 mg of each compound individually orin a single unit dosage form, such as from about 5 to about 300 mg, fromabout 10 to about 100 mg, and/or about 25 mg. The term “unit dosageform” refers to a physically discrete unit suitable as unitary dosagesfor a patient, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical carrier, diluent, orexcipient.

Oral administration is an illustrative route of administering thecompounds employed in the compositions and methods disclosed herein.Other illustrative routes of administration include transdermal,percutaneous, intravenous, intramuscular, intranasal, buccal,intrathecal, intracerebral, or intrarectal routes. The route ofadministration may be varied in any way, limited by the physicalproperties of the compounds being employed and the convenience of thesubject and the caregiver.

As one skilled in the art will appreciate, suitable formulations includethose that are suitable for more than one route of administration. Forexample, the formulation can be one that is suitable for bothintrathecal and intracerebral administration. Alternatively, suitableformulations include those that are suitable for only one route ofadministration as well as those that are suitable for one or more routesof administration, but not suitable for one or more other routes ofadministration. For example, the formulation can be one that is suitablefor oral, transdermal, percutaneous, intravenous, intramuscular,intranasal, buccal, and/or intrathecal administration but not suitablefor intracerebral administration.

The inert ingredients and manner of formulation of the pharmaceuticalcompositions are conventional. The usual methods of formulation used inpharmaceutical science may be used here. All of the usual types ofcompositions may be used, including tablets, chewable tablets, capsules,solutions, parenteral solutions, intranasal sprays or powders, troches,suppositories, transdermal patches, and suspensions. In general,compositions contain from about 0.5% to about 50% of the compound intotal, depending on the desired doses and the type of composition to beused. The amount of the compound, however, is best defined as the“effective amount”, that is, the amount of the compound which providesthe desired dose to the patient in need of such treatment. The activityof the compounds employed in the compositions and methods disclosedherein are not believed to depend greatly on the nature of thecomposition, and, therefore, the compositions can be chosen andformulated primarily or solely for convenience and economy.

Capsules are prepared by mixing the compound with a suitable diluent andfilling the proper amount of the mixture in capsules. The usual diluentsinclude inert powdered substances (such as starches), powdered cellulose(especially crystalline and microcrystalline cellulose), sugars (such asfructose, mannitol and sucrose), grain flours, and similar ediblepowders.

Tablets are prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants, and disintegrators (in addition to the compounds).Typical diluents include, for example, various types of starch, lactose,mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such assodium chloride), and powdered sugar. Powdered cellulose derivatives canalso be used. Typical tablet binders include substances such as starch,gelatin, and sugars (e.g., lactose, fructose, glucose, and the like).Natural and synthetic gums can also be used, including acacia,alginates, methylcellulose, polyvinylpyrrolidine, and the like.Polyethylene glycol, ethylcellulose, and waxes can also serve asbinders.

Tablets can be coated with sugar, e.g., as a flavor enhancer andsealant. The compounds also may be formulated as chewable tablets, byusing large amounts of pleasant-tasting substances, such as mannitol, inthe formulation. Instantly dissolving tablet-like formulations can alsobe employed, for example, to assure that the patient consumes the dosageform and to avoid the difficulty that some patients experience inswallowing solid objects.

A lubricant can be used in the tablet formulation to prevent the tabletand punches from sticking in the die. The lubricant can be chosen fromsuch slippery solids as talc, magnesium and calcium stearate, stearicacid, and hydrogenated vegetable oils.

Tablets can also contain disintegrators. Disintegrators are substancesthat swell when wetted to break up the tablet and release the compound.They include starches, clays, celluloses, algins, and gums. As furtherillustration, corn and potato starches, methylcellulose, agar,bentonite, wood cellulose, powdered natural sponge, cation-exchangeresins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, andcarboxymethylcellulose can be used.

Compositions can be formulated as enteric formulations, for example, toprotect the active ingredient from the strongly acid contents of thestomach. Such formulations can be created by coating a solid dosage formwith a film of a polymer which is insoluble in acid environments andsoluble in basic environments. Illustrative films include celluloseacetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and hydroxypropyl methylcellulose acetatesuccinate.

When it is desired to administer the compound as a suppository,conventional bases can be used. Illustratively, cocoa butter is atraditional suppository base. The cocoa butter can be modified byaddition of waxes to raise its melting point slightly. Water-misciblesuppository bases, such as polyethylene glycols of various molecularweights, can also be used in suppository formulations.

Transdermal patches can also be used to deliver the compounds.Transdermal patches can include a resinous composition in which thecompound will dissolve or partially dissolve; and a film which protectsthe composition and which holds the resinous composition in contact withthe skin. Other, more complicated patch compositions can also be used,such as those having a membrane pierced with a plurality of poresthrough which the drugs are pumped by osmotic action.

As one skilled in the art will also appreciate, the formulation can beprepared with materials (e.g., actives excipients, carriers (such ascyclodextrins), diluents, etc.) having properties (e.g., purity) thatrender the formulation suitable for administration to humans.Alternatively, the formulation can be prepared with materials havingpurity and/or other properties that render the formulation suitable foradministration to non-human subjects, but not suitable foradministration to humans.

The following list of formulations is illustrative. These illustrativeformulations may be suitable for preparing pharmaceutical compositionsthat include the disclosed compounds as “active ingredients.” Thefollowing list of formulations is illustrative and should not beinterpreted as limiting the present disclosure or claims in any way:

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

Quantity (mg/capsule) Active Ingredient 250 Starch, dried 200 Magnesiumstearate 10 Total 460 mgThe above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

Formulation 2

Quantity (mg/tablet) Active Ingredient 250 Cellulose, microcrystalline400 Silicon dioxide, fumed 10 Stearic acid 5 Total 665 mgThe components are blended and compressed to form tablets each weighing665 mg.

Formulation 3

An aerosol solution is prepared containing the following components:

Weight % Active Ingredient 0.25 Ethanol 29.75 Propellant 22(chlorodifluoromethane) 70.00 Total 100.00The active compound is mixed with ethanol and the mixture added to aportion of the Propellant 22, cooled to ⁻30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4

Tablets each containing 60 mg of active ingredient are made as follows:

Active Ingredient 60 mg Starch 45 mg Microcrystalline cellulose 35 mgPolyvinylpyrrolidone 4 mg Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1 mg Total 150 mgThe active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

Formulation 5

Capsules, each containing 80 mg medicament, are made as follows:

Active Ingredient 80 mg Starch 59 mg Microcrystalline cellulose 59 mgMagnesium stearate  2 mg Total 200 mg The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 sieve, and filled into hard gelatincapsules in 200 mg quantities.

Formulation 6

Suppositories each containing 225 mg of active ingredient may be made asfollows:

Active Ingredient   225 mg Saturated fatty acid glycerides 2,000 mgTotal 2,225 mgThe active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions each containing 50 mg of medicament per 5 ml dose are madeas follows:

Active Ingredient 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25ml Benzoic acid solution 0.10 ml Flavor q.v. Color q.v. Purified waterto total 5 mlThe medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl, cellulose and syrup to form a smooth paste.The benzoic acid solution, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation 8

An intravenous formulation containing 100 mg of medicament per 5 ml dosecan be prepared as follows:

Active Ingredient 100 mg Mannitol 100 mg 5N Sodium hydroxide 200 mlPurified water to total 5 ml

ILLUSTRATIVE EMBODIMENTS

The followings Embodiments are illustrative only and are not intended tolimit the scope of the claimed subject matter.

Embodiment 1. A compound or a salt or solvate thereof having a FormulaI:

wherein:

-   -   n is 0-3;    -   X is CH₂, NH, or O;    -   R¹ is selected from hydrogen, hydroxyl, alkyl, alkoxy, halo,        haloalkyl, amino, and cyano;    -   R² is hydrogen, or alkyl, or a 3-7 membered carbocycle or        heterocycle which is saturated or unsaturated at one or more        bonds and which heterocycle includes one or more heteroatoms        selected from N, O, and S, optionally which carbocycle or        heterocycle is substituted to include one or more non-hydrogen        substituents, which non-hydrogen substituents optionally are        selected from hydroxyl, alkyl, halo, haloalkyl, phenyl, amino,        and carbonyl.    -   R³ is present or absent, and when R³ is present, R³ is selected        from selected from hydrogen, alkyl, alkenyl, and alkyl-alkoxy.

Embodiment 2. The compound of embodiment 1 having Formula Ia:

Embodiment 3. The compound of embodiment 1 having a Formula Ib:

Embodiment 4. The compound of embodiment 1 having a Formula Ic:

Embodiment 5. The compound of embodiment 1 having a Formula Id:

Embodiment 6. The compound of embodiment 1 having a Formula Ie:

Embodiment 7. The compound of embodiment 1, wherein R¹ is selected fromhydrogen, alkyl, hydroxyl, alkyoxy, and halo.

Embodiment 8. The compound of embodiment 4, wherein R¹ is selected from:

Embodiment 9. The compound of embodiment 1, wherein R² is selected fromphenyl, cyclohexyl, pyridinyl, pyrimidinyl, imidazolyl,1-methylimidazolyl; piperidinyl, 1-methyl-piperidinyl, piperazinyl,1-methyl-piperazinyl, tetrahydropyranyl, and morpholinyl.

Embodiment 10. The compound of embodiment 1, wherein —(X)_(n)—R^(z) isselected from:

Embodiment 11. The compound of embodiment 5, wherein —(X)_(n)—R² isselected from:

Embodiment 12. The compound of any of the foregoing embodiments with theproviso that at least one of R¹, R², and R³ is not hydrogen.

Embodiment 13. The compound of embodiment 1 having a formula:

Embodiment 14. The compound of embodiment 1 having a formula selectedfrom:

Embodiment 15. A compound having a formula of any of the compounds ofembodiments 1-14 or a compound having a formula of any compounddisclosed in this application for use in treating and/or preventing adisease, disorder, or condition that is associated with 5-HT_(2c)receptor activity in a subject in need thereof.

Embodiment 16. A pharmaceutical composition comprising any of thecompounds of the foregoing embodiments and a pharmaceutical carrier.

Embodiment 17. A method for treating and/or preventing a disease,disorder, or condition that is associated with 5-HT_(2c) receptoractivity in a subject in need thereof, the method comprisingadministering to the subject the compound of any of embodiments 1-15 orthe pharmaceutical composition of embodiment 16.

Embodiment 18. The method of embodiment 17, wherein the disease ordisorder is a psychiatric, mental, or neurological disease, disorder, orcondition.

Embodiment 19. The method of embodiment 17, wherein the disease,disorder, or condition is cognitive impairment, addiction, and/orobsessive compulsive disorder (OCD).

Embodiment 20. The method of embodiment 17, wherein the disease,disorder, or condition is obesity and the method results in suppressingthe appetite of the subject.

Examples

The followings Examples are illustrative only and are not intended tolimit the scope of the claimed subject matter.

Beta-carbolines as Positive Allosteric Modulators of the Human SerotoninReceptor 2c (5-HT_(2c))

TECHNICAL FIELD

The technical field of the disclosed subject matter relates to smallmolecule drug development. The disclosed small molecules provide a drugplatform that has potential to treat cognitive impairment, addiction,obsessive compulsive disorder, and obesity via appetite suppression.

Abstract

We disclose novel small molecules having a substituted beta-carbolinecore. The disclosed small molecules have potential for treatingcognitive impairment (CI), for which there is currently no directtreatment. The disclosed small molecules also have potential fortreating addition. Although there are several drug options for treatingaddiction depending on the addiction, many are inadequate. For example,the current opioid epidemic exists despite the existence of treatmentoptions that include methadone, buprenorphinem and naltrexone. Thedisclosed small molecules also have potential for treating obsessivecompulsive disorder (OCD). OCD is most often treated with antidepressantmedications, but often patients do not respond to these medications.Electroconvulsive therapy can be tried in these cases, but this therapyis largely regarded as ineffective.

Applications

Applications of the disclosed small molecules include, but are notlimited to treatment of a range of neurological disorders and diseasesthat are associated with 5-HT₂, activity. Disorders and diseases thatmay be treated with the disclosed small molecules may include cognitiveimpairment (CI), addiction, and obsessive compulsive disorder (OCD). Thedisclosed small molecules also may suppress appetite and therefore maybe administered to treat obesity and eating disorders.

Advantages

There currently is only one FDA-approved drug that acts selectively on5-HT₂c, Lorcaserin, which currently is approved only for weight loss.However, Lorcaserin is associated with two dangerous side effects:cancer and cardiac valvulopathy. While Lorcaserin demonstratesselectivity for 5-HT₂c, Lorcaserin nonetheless exhibit activity for5-HT_(2a) and 5-HT_(2b).

Receptor EC₅₀ [nM] K_(i)[nM] 5-HT_(2C) 39 13 5-HT_(2B) 2380 1475-HT_(2A) 553 92

While Lorcaserin's selectivity for 5-HT₂, (K_(i)=39 nM) over 5-HT_(2a)(K_(i)=92 nM) and 5-HT_(2b) (K_(i)=147 nM) is good, selectivity can beimproved upon because positive allosteric modulators have been known tobind less conserved sites.

Brief Summary of the Technology

Small molecules have been designed having a substituted beta-carbolinecore and based on known yohimbine natural products alstonine andserpentine. The disclosed small molecuels exhibit in vivo activityconsistent with agonism or positive allosteric modulation of theserotonin receptor 5-HT₂c.

Compound Synthesis

General Procedure A: Pictet-Spengler reaction of serotonin-HCl andaldehydes to give tetrahydro-β-carbolines. To a solution of serotoninhydrochloride (1 equiv) in N,N-dimethylformamide (4.5 mL/mmol serotoninhydrochloride) was added the appropriate aldehyde (1.1 equiv). Theresulting solution was stirred at 60° C. overnight (16-20 hours). Afterthis time the reaction was allowed to cool to room temperature,neutralized with saturated sodium bicarbonate (equal volume toN,N-dimethylformamide), and was extracted with ethyl acetate (3×equalvolumes of the total mixture). The combined organic layers were washedwith additional water (1 equal volume), washed with saturated sodiumchloride (1 equal volume), dried over magnesium sulfate, filtered, andconcentrated to dryness. The resulting residue was purified via columnchromatography.

General Procedure B: Palladium on carbon aromatization oftetrahydro-p-carbolines to give β-carbolines. To a flask containing drytetrahydro-β-carboline (1 equiv) and palladium on carbon (10 wt %, 0.1equiv) was added anisole (15 mL/mmol of tetrahydro-p-carboline). Theresulting mixture was heated at reflux overnight (16-20 hours). If thereaction had not proceeded sufficiently, minimal amounts of methanolwere added to aid with solubility, and the reaction was resubmitted tothe previous conditions. After this time the reaction was allowed tocool to room temperature, filtered through Celite® (rinsing withmethanol), and concentrated to dryness. The resulting residue waspurified via column chromatography.

General Procedure C: Alkylation of β-carbolines to give N-alkylβ-carbolines. To a solution of β-carboline (1 equiv) inN,N-dimethylformamide (7 mL/mmol of β-carboline) was added theappropriate alkyl halide (1.1 equiv). The resulting mixture was heatedat 85° C. overnight (16-20 hours). After this time the reaction wasallowed to cool to room temperature. If starting material was notconsumed, additional alkyl halide would be added and resubmitted to theprevious conditions. After this time, the reaction was concentrated todryness. The resulting residue was purified via column chromatography.

1-phenyl-1H,2H,3H,4H,9H-pyrido[3,4-b]indol-6-ol was synthesizedaccording to general procedure A and was purified on 10 g silica gel,with a gradient of dichloromethane to 6% methanol/94% dichloromethane togive the title product (0.120 g, 32%) as an off-white solid; H NMR (500MHz, DMSO-d₆) δ 10.04 (s, 1H), 8.51 (s, 1H), 7.37-7.21 (m, 6H), 7.16(dd, J=18.3, 7.3 Hz, 1H), 6.99 (d, J=8.5 Hz, 1H), 6.72 (s, 1H), 6.50(dd, J=8.5, 2.2 Hz, 1H), 5.02 (s, 1H), 3.05-3.01 (m, 1H), 2.93-2.88 (m,1H), 2.67-2.62 (m, 1H), 2.59-2.54 (m, 1H). ¹³C NMR (126 MHz, DMSO) δ150.15, 143.31, 135.85, 130.36, 128.38, 128.03, 127.51, 127.07, 111.25,110.36, 107.43, 101.82, 56.65, 41.25, 21.05. MS (ESI) calculated forC₁₇H₁₇N₂O [M+H]⁺: 265.1, Found: 265.2.

1-phenyl-9H-pyrido[3,4-b]indol-6-ol was synthesized according to generalprocedure B and was purified on 10 g silica gel, with a gradient of [10g silica, dichloromethane to 4% methanol/96% dichloromethane to give thetitle product (0.359 g, 53%) as a tan solid; ¹H NMR (500 MHz, DMSO-d₆) δ11.18 (s, 1H), 9.15 (s, 1H), 8.37 (d, J=5.1 Hz, 1H), 8.00 (dd, J=10.9,6.4 Hz, 3H), 7.60 (t, J=7.5 Hz, 2H), 7.55-7.42 (m, 3H), 7.07 (dd, J=8.7,2.2 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 151.03, 141.96, 138.53, 137.56,137.34, 135.17, 133.55, 128.83, 128.66, 128.38, 128.26, 121.43, 118.26,112.96, 105.41. MS (ESI) calculated for C₁₇H₁₃N₂O [M+H]⁺: 261.1, Found:262.1.

6-hydroxy-1-phenyl-2-(prop-2-en-1-yl)-9H-pyrido[3,4-b]indol-2-iumbromide was synthesized according to general procedure C and waspurified on 25 g silica gel, with a gradient of dichloromethane to 7%methanol/93% dichloromethane to give the title product (0.143 g, 64%) asa dark yellow solid; ¹H NMR (500 MHz, DMSO-d₆) δ 11.98 (s, 1H), 9.69 (s,1H), 8.80 (d, J=6.6 Hz, 1H), 8.69 (d, J=6.6 Hz, 1H), 7.76 (m, 6H), 7.53(d, J=8.9 Hz, 1H), 7.33 (dd, J=8.9, 2.4 Hz, 1H), 6.20-5.73 (m, 1H), 5.26(d, J=10.5 Hz, 1H), 5.08 (d, J=5.2 Hz, 2H), 4.95 (d, J=17.2 Hz, 1H). ¹³CNMR (126 MHz, DMSO) δ 152.55, 140.09, 139.09, 135.27, 132.53, 132.43,131.74, 131.35, 129.66, 129.40, 128.23, 123.16, 120.33, 119.23, 117.22,113.92, 105.91, 58.69. MS (ESI) calculated for C₂₀H₁₇N₂O [M]⁺: 301.1,Found: 301.3.

6-hydroxy-2-(3-methoxypropyl)-1-phenyl-9H-pyrido[3,4-b]indol-2-iumbromide was synthesized according to general procedure C and waspurified on 10 g silica gel, with a gradient of dichloromethane to 20%methanol/80% dichloromethane to give the title product (0.045 g, 28%) asa dark brown solid; ¹H NMR (500 MHz, DMSO-d₆) δ 11.90 (s, 1H), 9.67 (s,1H), 8.78-8.73 (m, 2H), 7.79-7.74 (q, J=17.8, 11.1 Hz, 6H), 7.52 (d,J=8.9 Hz, 1H), 7.32 (dd, J=8.9, 2.3 Hz, 1H), 4.49 (t, J=7.4 Hz, 2H),3.22 (t, J=5.8 Hz, 2H), 3.06 (s, 3H), 1.98 (p, J=6.1 Hz, 2H). ¹³C NMR(126 MHz, DMSO) δ 152.51, 139.98, 138.91, 135.27, 132.50, 131.47,131.25, 129.69, 129.51, 128.38, 123.00, 120.30, 117.09, 113.88, 105.91,68.16, 57.68, 54.57, 30.32. MS (ESI) calculated for C₂₁H₂₁N₂O₂ [M]⁺:333.2, Found: 333.3.

1-cyclohexyl-1H,2H,3H,4H,9H-pyrido[3,4-b]indol-6-ol was synthesizedaccording to general procedure A and was purified on 10 g silica gel,with a gradient of dichloromethane to 20% methanol/80% dichloromethaneto give the title product (0.083 g, 22%) as a tan solid; ¹H NMR (500MHz, DMSO-d₆) δ 10.66 (s, 1H), 8.70 (s, 1H), 7.48-6.95 (m, 1H), 6.73 (s,1H), 6.62-6.60 (m, 1H), 4.43 (s, 1H), 3.48-3.46 (m, 1H), 3.25-3.15 (m,1H), 2.98-2.73 (m, 2H), 2.20-2.10 (m, 1H), 1.82-1.65 (m, 4H), 1.38-1.10(m, 7H). ¹³C NMR (126 MHz, DMSO) δ 150.65, 130.66, 130.06, 126.69,111.64, 111.57, 105.93, 101.93, 57.36, 41.84, 29.23, 26.66, 26.08,25.71, 18.58. MS (ESI) calculated for C₁₇H₂₃N₂O [M+H]⁺: 271.2, Found:271.0.

1-cyclohexyl-9H-pyrido[3,4-b]indol-6-ol was synthesized according togeneral procedure B and was purified on 50 g silica gel, with a gradientof dichloromethane to acetone to give the title product (0.093 g, 31%)as a beige solid; ¹H NMR (500 MHz, DMSO-d₆) δ 11.17 (s, 1H), 9.06 (s,1H), 8.15 (d, J=5.2 Hz, 1H), 7.77 (d, J=5.2 Hz, 1H), 7.44 (d, J=2.2 Hz,1H), 7.40 (d, J=8.7 Hz, 1H), 7.04 (dd, J=8.7, 2.4 Hz, 1H), 3.28-3.20 (m,1H), 1.91-1.82 (m, 2H), 1.81-1.66 (m, 3H), 1.56-1.41 (m, 2H), 1.39-1.25(m, 1H). ¹³C NMR (126 MHz, DMSO) δ 150.68, 149.74, 136.68, 134.39,133.78, 126.82, 121.75, 117.77, 112.38, 112.37, 105.43, 40.80, 31.31,26.24, 25.84. MS (ESI) calculated for C₁₇H₁₉N₂O [M+H]⁺: 267.2, Found:267.4.

1-cyclohexyl-6-hydroxy-2-(prop-2-en-1-yl)-9H-pyrido[3,4-b]indol-2-iumbromide was synthesized according to general procedure C and waspurified on 10 g silica gel, with a gradient of dichloromethane toacetone to give the title product (0.066 g, 46%) as a dark yellow solid;¹H NMR (500 MHz, DMSO-d₆) δ 12.00 (s, 1H), 9.65 (s, 1H), 8.63 (d, J=6.5Hz, 1H), 8.56 (d, J=6.2 Hz, 1H), 7.73 (d, J=8.9 Hz, 1H), 7.68 (d, J=2.2Hz, 1H), 7.36 (dd, J=8.9, 2.4 Hz, 1H), 6.25-6.17 (m, 1H), 5.51 (s, 2H),5.36 (d, J=10.6 Hz, 1H), 5.02 (d, J=17.3 Hz, 1H), 3.50-3.39 (m, 1H),2.35-2.16 (m, 2H), 2.01-1.62 (m, 6H), 1.53-1.40 (m, 2H). ¹³C NMR (126MHz, DMSO) δ 152.51, 146.40, 138.08, 133.81, 133.21, 133.15, 132.32,123.06, 119.76, 118.00, 116.14, 113.94, 105.56, 59.72, 48.58, 27.96,25.71, 23.96. MS (ESI) calculated for C₂₀H₂₃N₂O [M]⁺: 307.2, Found:307.3.

1-(pyrimidin-5-yl)-1H,2H,3H,4H,9H-pyrido[3,4-b]indol-6-ol wassynthesized according to general procedure A and was purified on 10 gsilica gel, with a gradient of dichloromethane to 10% methanol/90%dichloromethane to give the title product (0.137 g, 37%) as an off-whitesolid; ¹H NMR (500 MHz, DMSO-d₆) δ 10.23 (s, 1H), 9.12 (s, 1H), 8.67 (s,2H), 8.58 (s, 1H), 7.02 (d, J=8.5 Hz, 1H), 6.75 (d, J=2.0 Hz, 1H), 6.55(dd, J=8.5, 2.2 Hz, 1H), 5.14 (s, 1H), 3.14-2.92 (m, 3H), 2.74-2.64 (m,1H), 2.64-2.55 (m, 2H). ¹³C NMR (126 MHz, DMSO) δ 157.33, 156.69,150.36, 136.39, 134.06, 130.45, 127.44, 111.35, 110.84, 108.06, 102.01,52.39, 41.38, 22.03. MS (ESI) calculated for C₁₅H₁₅N₄O [M+H]⁺: 267.1,Found: 267.2.

1-(pyrimidin-5-yl)-9H-pyrido[3,4-b]indol-6-ol (AJC-61) was synthesizedaccording to general procedure B and was purified on 25 g silica gel,with a gradient of dichloromethane to 50% acetone/50% dichloromethane togive the title product (0.024 g, 5%) as a yellow solid; ¹H NMR (500 MHz,DMSO-d₆) δ 11.54 (s, 1H), 9.40 (s, 2H), 9.32 (s, 1H), 9.23 (s, 1H),8.51-8.40 (m, 1H), 8.19-8.06 (m, 1H), 7.57 (s, 1H), 7.47 (d, J=8.7 Hz,1H), 7.11 (d, J=8.6 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ 157.83, 156.08,151.29, 138.05, 136.13, 135.29, 134.04, 132.02, 129.36, 121.28, 118.76,115.10, 112.96, 105.64. MS (ESI) calculated for C₁₅H₁₁N₄O [M+H]⁻: 263.1,Found: 263.1.

TABLE 1 Activity of Selected Compounds.

AJC-61 2C agonist inactive antagonist >10 μM 2A agonist inactiveantagonist no data

MJO-01-31 2C agonist 10.1 nM Emax 101% 2A agonist 54.6 nM Emax  95%

MJO-01-34 2C agonist 85.9 nM Emax 103% 2A agonist 106 nM Emax  27%

MJO-01-46 2C agonist 5.37 nM Emax  98% 2A agonist 11.3 nM Ernax  96%

Biological Activity of AJC-61

Mouse models were utilized to demonstrate the potential of compoundAJC-61 in treating and or affecting psychotic spectrumdisorder-schizophrenia including cognitive impairment of schizophrenia,negative symptoms of schizophrenia including deficit in socialinteraction, bipolar disorder, major depression including psychoticmajor depression, locomotor activity suppression-specific for positive(psychotic) symptoms of schizophrenia, psychoses of Alzheimer's diseaseand Parkinson's disease, obsessive compulsive disorder, Tourette'ssyndrome, and age-associated cognitive impairment.

The ability of a test drug to block the increase in Locomotor Activity(LMA) produced by amphetamine or the glutamate receptor agonist,phencyclidine (PCP), has a high predictive value for the antipsychoticefficacy of the test drug for treating schizophrenia. Administration ofamphetamine or PCP to man has been shown to produce psychotic state in asignificant percentage of previously normal humans. Treatment of micewith PCP for one week has been shown to produce a robust model ofschizophrenia. As such, we tested the effect of AJC-61 on reducing LMAin mice that had been treated with amphetamine or PCP to increase LMAactivity. As shown in FIG. 1 , AJC-61 blocked the amphetamine-inducedincrease in LMA in mice, and as shown in FIG. 2 , AJC-61 blocked thePCP-induced increase in LAM in mice.

The Novel Object Recognition test in rodents is considered a valid modelof spatial memory in man. The time spent viewing an object seenpreviously versus the time spent viewing a novel object (i.e., theDiscrimination Index (DI)) provides an indirect measure of assessingretention of the memory of the object that had previously been shown tothe rodent. Normal mice remember for ˜24 hours. The time to loss of thememory in aged mice can be as low as zero to less than 8 hours. As such,we tested the effect of AJC-61 on reducing the DI in mice that had beentreated with PCP to reduce the DI. As shown in FIG. 3 , we observed asignificant increase in DI in the PCP+AJC-61—treated mice versus thePCP-treated mice.

A healthy mouse will vigorously explore a newly introduce mouse andretain the memory for that mouse for up to 24 hours. The loss ofinterest in exploring a novel mouse is considered a sign of negativesymptoms.

PCP-treatment produces a deficit in social interaction which modelsnegative symptoms in schizophrenia. These include lack of motivation,loss of interest in activities, diminished capacity for pleasure andspontaneous activity. We tested the effect of AJC-61 on SocialInteraction in mice that had been treated with PCP, including a SocialBehaviors that include sniffing, following, and avoiding and ObjectExploration via interaction with an inanimate object. As illustrated inFIG. 4 , regarding sniffing we observed a significant reduction insniffing for the PCP group versus the Vehicle group, and a significantincrease in sniffing for the PCP+AJC-61 versus group versus the PCPgroup. Regarding following, we observed no significant change, althoughthere was non-significant reduction in following in the PCP group versusthe Vehicle group. Regarding avoiding, we observed a significantincrease for the PCP versus group versus the Vehicle group, and asignificant decrease in avoiding for the PCP+AJC-61 group versus the PCPgroup. Regarding object exploration, we observed no significant change,although there was a non-significant increase in object exploration inthe PCP group versus the Vehicle group.

The Porsolt Forced Swim Test (FST) is a gold standard test forantidepressant action. In the FST, normal mice become immobile afterbeing required to swim in a tank and begin to float instead ofstruggling to get out of the water. Antidepressant drugs have been shownto increase the amount of time that a mouse will continue to swim priorto floating. As such, we tested whether AJC-61 could increase the swimtime in a FST. As illustrated in FIG. 5 , treatment with PCPsignificantly increased the immobility time whereas treatment withPCP+AJC-61 significantly decreased the immobility time.

Marble Burying (MB) and extent of shredding a standard type of rodentbedding (i.e., Nestlet Shredding) are both proven methods for assessingdrug efficacy to treat obsessive-compulsive disorder and Tourette'ssyndrome. As such, we tested the effect on AJC-61 on Marble Burying andNestlet Shredding. As illustrated in FIG. 6 , we observed a significantincrease in the number of marbles buried for the PCP group versus theVehicle group, and we observed a significant decrease in the number ofmarbles buried for the PCP+AJC-16 group versus the PCP group. Asillustrated in FIG. 7 , we observed a significant increase in thepercent nestlet shredded for the PCP group versus the Vehicle group, andwe observed a significant decrease in the percent nestlet shredded forthe PCP+AJC-16 group versus the PCP group.

Aged mice show a decline in memory function beginning around 13-15 monthof age. Drugs which enhance memory can be assessed using the NovelObject Recognition test and determination of the Discrimination Index(DI) as discussed above. As such, we tested whether AJC-61 could improvethe DI for aged mice (i.e., 22-month old mice) versus young mice (i.e.,2.5 month old mice). As illustrated in FIG. 8 , we observed asignificant decrease in the DI for 22-month old mice versus the DI for2.5 month old mice, and we observed a significant increase in the DI for22-month old mice when the mice were treated with AJC-61.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein. The terms and expressions whichhave been employed are used as terms of description and not oflimitation, and there is no intention in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenillustrated by specific embodiments and optional features, modificationand/or variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

1.-20. (canceled)
 21. A compound or a salt or solvate thereof having aFormula I:

wherein: n is 0-3; X is CH₂, NH, or O; R¹ is selected from hydrogen,hydroxyl, alkyl, alkoxy, halo, haloalkyl, amino, and cyano; R² ishydrogen, or alkyl, or a 3-7 membered carbocycle or heterocycle which issaturated or unsaturated at one or more bonds and which heterocycleincludes one or more heteroatoms selected from N, O, and S, optionallywhich carbocycle or heterocycle is substituted to include one or morenon-hydrogen substituents, which non-hydrogen substituents optionallyare selected from hydroxyl, alkyl, halo, haloalkyl, phenyl, amino, andcarbonyl. R³ is present or absent, and when R³ is present, R³ isselected from selected from hydrogen, alkyl, alkenyl, and alkyl-alkoxy.22. The compound of claim 21, wherein R² is selected from phenyl,cyclohexyl, pyridine-2-yl, pyridine-3-yl, imidazolyl,1-methylimidazolyl; piperidinyl, 1-methyl-piperidinyl, piperazinyl,1-methyl-piperazinyl, tetrahydropyranyl, and morpholinyl.
 23. Thecompound of claim 21, wherein —(X)_(n)—R² is selected from:


24. The compound of claim 21, wherein —(X)_(n)—R² is selected fromphenyl and cyclohexyl.
 25. The compound of claim 21, wherein thecompound is selected from:


26. The compound of claim 21 having formula Ib:


27. The compound of claim 26, wherein R² is selected from phenyl,cyclohexyl, pyridin-2-yl, pyridin-3-yl, imidazolyl, 1-methylimidazolyl;piperidinyl, 1-methyl-piperidinyl, piperazinyl, 1-methyl-piperazinyl,tetrahydropyranyl, and morpholinyl.
 28. The compound of claim 26,wherein —(X)_(n)—R² is selected from:


29. The compound of claim 21, wherein R¹ is selected from hydroxyl,halo, methyl, and hydrogen.
 30. The compound of claim 29, wherein R² isselected from phenyl, cyclohexyl, pyridinyl, imidazolyl,1-methylimidazolyl; piperidinyl, 1-methyl-piperidinyl, piperazinyl,1-methyl-piperazinyl, tetrahydropyranyl, and morpholinyl.
 31. Thecompound of claim 29, wherein —(X)_(n)—R² is selected from:


32. The compound of claim 29 having formula Ib:


33. The compound of claim 32, wherein R² is selected from phenyl,cyclohexyl, pyridinyl, imidazolyl, 1-methylimidazolyl; piperidinyl,1-methyl-piperidinyl, piperazinyl, 1-methyl-piperazinyl,tetrahydropyranyl, and morpholinyl.
 34. The compound of claim 32,wherein —(X)_(n)—R² is selected from:


35. The compound of claim 32, wherein R¹ is hydroxyl.
 36. Apharmaceutical composition comprising the compound of claim 21 and apharmaceutical carrier.
 37. A method for treating and/or preventing adisease, disorder, or condition that is associated with 5-HT_(2c)receptor activity in a subject in need thereof, the method comprisingadministering to the subject the compound of claim
 21. 38. The method ofclaim 37, wherein the disease or disorder is a psychiatric, mental, orneurological disease, disorder, or condition.
 39. The method of claim37, wherein the disease, disorder, or condition is cognitive impairment,addiction, and/or obsessive compulsive disorder.
 40. The method of claim37, wherein the disease, disorder, or condition is obesity and themethod results in suppressing the appetite of the subject.